ABSTRACT In the US, an estimated 1.2 million people are living with HIV. In spite of considerable progress, anti- retroviral therapy (ART) remains the only treatment option. The long-term success of ART is dependent on identification of new drugs against novel therapeutic due to toxicity of current drugs and emergence of ART- resistant viruses. The anti-HIV activity of cellular miRNAs is an emerging area and has a great potential to identify novel therapeutic targets. However, very little is known about the molecular interactions between cellular miRNAs and HIV-1. MiRNAs are small noncoding RNAs that negatively regulate gene expression by binding to the 3? untranslated region (UTR) of target mRNAs causing translational repression or mRNA degradation. miRNAs play fundamental roles in every aspect of cellular process including anti-viral responses. Published data demonstrate that cellular miRNAs regulate replication of many viruses including Primate foamy virus, Hepatitis C virus, Epstein Barr virus, Influenza virus and HIV. The overall goal of this application is to dissect the mechanism by which cellular miRNAs confer anti-HIV activity. The anti-HIV-1 activity of cellular miRNAs was first discovered by target prediction coupled with experimental validation. Since then a family of anti-HIV miRNAs has been reported to inhibit HIV-1. Two mechanisms are proposed for the anti-HIV activities of cellular miRNAs. They can bind to HIV-1 transcripts and inhibit viral protein translation. Alternatively, they can bind to cellular transcripts and inhibit translation of host factors associated with viral replication. However, there are key knowledge gaps in the understanding of miRNA-mediated anti-HIV activity. Our proposed studies on the cellular anti-HIV-1 miRNAs are designed to fill this gap. The central hypothesis is anti-HIV-1 miRNAs inhibit HIV-1 by repressing viral protein translation and regulation of anti-HIV miRNAs is dependent on DNMT-assisted promoter hyper- methylation. This proposal is organized to test the central hypothesis through three specific aims. Aim 1: To determine that repression of HIV-1 translation is mediated by direct binding of anti-HIV miRNAs to viral transcripts. Aim 2: To test that DNMT-induced promoter hyper-methylation regulates anti-HIV miRNA expression. Aim 3: To elucidate the mechanism by which CpG hyper-methylation causes downregulation of anti-HIV-1 miRNAs. The approach used in this application utilizes novel methods at the interface of virology, genetics, biochemistry, and molecular biology. We strongly believe that data from this proposal will significantly improve our understanding of anti-HIV-1 activity of cellular miRNAs and facilitate development of novel anti- viral therapy.